The present invention relates generally the regulation of apoptosis, and more particularly, to truncated Apaf-1 and methods of using truncated Apaf-1 and self-oligomerizing caspases to identify modulators of apoptosis.
Apoptosis is the normal physiological process of programmed cell death that maintains tissue homeostasis. Changes to the apoptotic pathway that prevent or delay normal cell turnover can be just as important in the pathogenesis of diseases as are abnormalities in the regulation of the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either prevent or induce cell death.
Since apoptosis functions in maintaining tissue homeostasis in a range of physiological processes such as embryonic development, immune cell regulation and normal cellular turnover, the dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes that occurs with many autoimmune diseases. Inappropriate loss or inhibition of apoptosis can also lead to the accumulation of virally infected cells and of hyperproliferative cells such as neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can also contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. Treatments which are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can alter the natural progression of many of these diseases.
Although apoptosis is mediated by diverse signals and complex interactions of cellular gene products, the results of these interactions ultimately feed into a cell death pathway that is evolutionarily conserved between humans and invertebrates. The pathway, itself, is a cascade of proteolytic events analogous to that of the blood coagulation cascade.
Several gene families and products that modulate the apoptotic process have now been identified. One family is the aspartate-specific cysteine proteases (xe2x80x9ccaspasesxe2x80x9d). The human caspase family includes, for example, Ced-3, human ICE (interleukin-1-xcex2 converting enzyme) (caspase-1), ICH-1 (caspase-2), CPP32 (caspase-3), ICErelII (caspase-4), ICErelIII (caspase-5), Mch2 (caspase-6), ICE-LAP3 (casepase-7), Mch5 (caspase-8), ICE-LAP6 (caspase-9), Mch4 (caspase-10), and others.
The caspase proteins share several common features. In this regard, caspases are cysteine proteases (named for a cysteine residue in the active site) that cleave substrates at Asp-X bonds. Furthermore, caspases are primarily produced as inactive zymogens that require proteolytic cleavage at specific internal aspartate residues for activation. The primary gene product is arranged such that the N-terminal peptide (prodomain) precedes a large subunit domain, which precedes a small subunit domain. The large subunit contains the conserved active site pentapeptide QACXG (X=R, Q, G) which contains the nucleophilic cysteine residue. The small subunit contains residues that bind the Asp carboxylate side chain and others that determine substrate specificity. Cleavage of a caspase yields the two subunits, the large (generally approximately 20 kD) and the small (generally approximately 10 kD) subunit that associate non-covalently: to form a heterodimer, and, in some caspases, an N-terminal peptide of varying length. The heterodimer may combine non-covalently to form a tetramer.
Caspase zymogens are themselves substrates for caspases. Inspection of the interdomain linkages in each zymogen reveals target sites (i.e. protease sites) that indicate a hierarchical relationship of caspase activation. By analyzing such pathways, it has been demonstrated that caspases are required for apoptosis to occur. Moreover, caspases appear to be necessary for the accurate and limited proteolytic events which are the hallmark of classic apoptosis (see Salvesen and Dixit, Cell 91:443-446, 1997). Once activated, most caspases can process and activate their own and other inactive procaspases in vitro (Fernandes-Alnemri et al., Proc. Natl. Acad. Sci. USA 93:7464-7469, 1996; Srinivasula et al., Proc. Natl. Acad. Sci. USA 93:13706-13711, 1996. This characteristic suggests that caspases implicated in apoptosis may execute the apoptotic program through a cascade of sequential activation of initiators and executioner procaspases (Salvesen and Dixit, Cell 91:443-446, 1997). The initiators are responsible for processing and activation of the executioners. The executioners are responsible for proteolytic cleavage of a number of cellular proteins leading to the characteristic morphological changes and DNA fragmentation that are often associated with apoptosis (reviewed by (Cohen, Biochem. J. 326:1-16, 1997; Henkart, Immunity 4:195-201, 1996; Martin and Green, Cell 82:349-352, 1995; Nicholson and Thomberry, TIBS 257:299-306, 1997; Porter et al., BioEssays 19:501-507, 1997; Salvesen and Dixit, Cell 91:443-446, 1997. The first evidence for an apoptotic caspase cascade was obtained from studies on death receptor signaling (reviewed by (Fraser and Evan, Cell 85:781-784, 1996; Nagata, Cell 88:355-365, 1997) which indicated that the death signal is transmitted in part by sequential activation of the initiator procaspase-8 and the executioner procaspase-3 (Boldin et al., Cell 85:803-815, 1996; Fernandes-Alnemri et al., Proc. Natl. Acad. Sci. USA 93:7464-7469, 1996; Muzio et al., Cell 85:817-827, 1996; Srinivasula et al., Proc. Natl. Acad. Sci. USA 93:13706-13711, 1996). More direct evidence was provided, recently, when it was demonstrated that the cytochrome c death signal is transmitted through activation of a cascade involving procaspase-9 and -3 (Li et al., Cell 91:479-489, 1997).
However, it remains unclear how the initiator caspases, like procaspase-8 and -9 are activated. While Apaf-1 is known to play a role in the activation of procaspase-9 the exact mechanism has yet to be determined.
Therefore, there exists a need in the art for methods of assaying compounds for their ability to affect Apaf-1 mediated caspase activity as well as for methods of modulating apoptosis in order to treat diseases and syndromes. The present invention fulfills this need, while further providing other related advantages.
The present invention generally provides truncated Apaf-1. In one aspect, the invention provides an isolated nucleic acid molecule encoding a truncated Apaf-1 or a variant thereof. In one embodiment, the encoded truncated Apaf-1 is a human truncated Apaf-1. In another embodiment, the human truncated Apaf-1 has the amino acid sequence of SEQ ID NO:2 or a variant thereof. In another embodiment, the nucleic acid molecule encoding a truncated Apaf-1 or variant thereof has the nucleic acid sequence of SEQ ID NO:1 or a variant thereof. In another embodiment, the nucleic acid molecule encodes a truncated Apaf-1 or fragment thereof that oligomerizes with a caspase. In yet another embodiment, the nucleic acid molecule encodes a human truncated Apaf-1 having the amino acid sequence of SEQ ID NO:2 or variant thereof that oligomerizes with a caspase.
It is another aspect of the invention to provide an expression vector comprising any of the nucleic acid molecules encoding a truncated Apaf-1 or a variant thereof referred to above, wherein the nucleic acid molecule encoding the truncated Apaf-1 is operatively linked to a promoter. In one embodiment, the promoter is inducible. In another aspect, the invention provides a host cell transfected with such expression vectors. In certain embodiments, the host cell may be a bacterium, an insect cell or a mammalian cell.
Another aspect of the invention pertains to an isolated truncated Apaf-1 polypeptide or fragment thereof. In one embodiment, the isolated truncated Apaf-1 polypeptide or fragment thereof oligomerizes with a caspase. In another embodiment, the isolated truncated Apaf-1 polypeptide or fragment thereof is a human truncated Apaf-1, which in a further embodiment may oligomerize with a caspase. In certain embodiments, the caspase with which an isolated truncated Apaf-1 polypeptide or fragment thereof oligomerizes may be caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12 or caspase-13. In certain embodiments, the caspase with which an isolated human truncated Apaf-1 polypeptide or fragment thereof oligomerizes may be caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, procaspase-9, caspase-10, caspase-11, caspase-12 or caspase-13. In another embodiment, the isolated human truncated Apaf-1 polypeptide or fragment thereof comprises SEQ ID NO:2 or a variant thereof. In another embodiment, the isolated human truncated Apaf-1 polypeptide or fragment thereof is encoded by SEQ ID NO:1 or a variant thereof.
It is another aspect of the invention to provide a method of identifying an inhibitor or enhancer of Apaf-1 mediated caspase processing, by contacting a sample containing a truncated Apaf-1 or fragment thereof and one or more caspases with a candidate inhibitor or candidate enhancer, detecting the presence of large and small caspase subunits, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase processing inhibitor, and wherein an increase in processing indicates the presence of a caspase processing enhancer. In one embodiment of this aspect of the invention, at least one of the caspases is procaspase-9 or a functional fragment thereof. In another embodiment, the caspase is in vitro translated and labeled. In a further embodiment, the label may be a radioactive label, a peptide tag, an enzyme or biotin.
Another aspect of the invention provides a method of identifying an inhibitor or enhancer of Apaf-1 mediated caspase processing, by contacting a cell transfected with a vector expressing a nucleic acid encoding truncated Apaf-1 or a variant thereof as described above with a candidate inhibitor or candidate enhancer, detecting the presence of large and small caspase subunits, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase processing enhancer.
In certain embodiments of these methods of identifying an inhibitor or enhancer of Apaf-1 mediated caspase processing, the step of detecting comprises gel electrophoresis.
Turning to another aspect of the invention, a method is provided for identifying an inhibitor or enhancer of Apaf-1 mediated apoptosis by contacting a cell transfected with a vector expressing truncated Apaf-1 or a variant thereof as described above with a candidate inhibitor or candidate enhancer, and detecting cell viability, wherein an increase in cell viability indicates the presence of an inhibitor and an decrease in cell viability indicates an enhancer.
It is another aspect of the invention to provide a method for inducing apoptosis in a cell, by delivering to a cell an effective amount of a nucleic acid molecule encoding a truncated Apaf-1 polypeptide, and maintaining the cell under conditions sufficient for expression of the polypeptide. In one embodiment, the step of delivering comprises injecting the nucleic acid molecule into the cell. In another embodiment, the step of delivering comprises administering the nucleic acid molecule to the circulatory system of a warm-blooded mammal in which the cell is located.
In another aspect of the invention, an antisense nucleic acid molecule is provided comprising a nucleic acid sequence that is complementary to a nucleic acid molecule encoding a truncated Apaf-1.
In another aspect of the invention, a gene delivery vehicle is provided comprising a nucleic acid molecule encoding truncated Apaf-1 or a variant thereof as described above wherein the nucleic acid molecule is operatively linked to a promoter. In certain embodiments the vehicle is a retrovirus or adenovirus. In certain other embodiments, the nucleic acid molecule is associated with a polycation. In certain other embodiments, the gene delivery vehicle may further comprise a ligand that binds a cell surface receptor.
The invention also provides a method of treating cancer, by administering to a patient a gene delivery vehicle comprising a nucleic acid molecule encoding truncated Apaf-1 or a variant thereof as described above, wherein the nucleic acid molecule is operatively linked to a promoter and wherein the gene delivery vehicle is internalized by tumor cells.
In another aspect the invention provides a method of treating autoimmune disease by administering to a patient a gene delivery vehicle comprising a nucleic acid molecule encoding truncated Apaf-1 or a variant thereof as described above wherein the nucleic acid molecule is operatively linked to a promoter and which may further be a retrovirus or adenovirus, a nucleic acid molecule associated with a polycation or a ligand that binds a cell surface receptor, and wherein the gene delivery vehicle is internalized by cells mediating autoimmune disease.
Another aspect of the invention provides a method of identifying an inhibitor of Apaf-1 mediated caspase processing by contacting a cell transfected with an inducible expression vector expressing truncated Apaf-1 or a variant thereof as described above with a candidate inhibitor; contacting the transfected cell with an inducer capable of inducing truncated Apaf-1 expression, and detecting the presence of large and small caspase subunits, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase processing inhibitor. In one embodiment, the step of detecting comprises gel electrophoresis.
In another aspect of the invention, a method is provided for identifying an inhibitor of Apaf-1 mediated apoptosis by contacting a cell transfected with an inducible expression vector expressing truncated Apaf-1 or a variant thereof as described above with a candidate inhibitor, contacting the transfected cell with an inducer capable of inducing truncated Apaf-1 expression; and detecting cell viability, wherein an increase in cell viability indicates the presence of an inhibitor.
Another aspect of the invention provides a method of identifying an inhibitor of apoptosis by contacting a cell transfected with an inducible expression vector capable of expressing a self-oligomerizing and self-processing caspase with a candidate inhibitor; contacting the transfected cell with an inducer capable of inducing self-oligomerizing caspase expression, and detecting the presence of large and small caspase subunits, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of an apoptosis inhibitor. In one embodiment, the step of detecting comprises gel electrophoresis.
Another aspect of the invention provides a method of identifying an inhibitor of apoptosis, by contacting a cell transfected with an inducible expression vector capable of expressing a self-oligomerizing and self-processing caspase with a candidate inhibitor; contacting the transfected cell with an inducer capable of inducing self-oligomerizing caspase expression; and detecting cell viability, wherein an increase in cell viability indicates the presence of an inhibitor.
Another aspect of the invention provides a method for inhibiting apoptosis in a cell by delivering to a cell an effective amount of a nucleic acid molecule encoding a caspase-9 prodomain; and maintaining the cell under conditions sufficient for expression of the polypeptide.
Another aspect of the invention provides a method for inducing apoptosis in a cell, by delivering to a cell an effective amount of a nucleic acid molecule encoding a self-oligomerizing caspase-9 polypeptide; and maintaining the cell under conditions sufficient for expression of the polypeptide. In one embodiment, the step of delivering comprises injecting the nucleic acid molecule into the cell. In another embodiment, the step of delivering comprises administering the nucleic acid molecule to the circulatory system of a warm-blooded mammal in which the cell is located.
Another aspect of the invention provides a gene delivery vehicle comprising a nucleic acid molecule encoding a self-oligomerizing caspase-9 polypeptide, wherein the nucleic acid molecule is operatively linked to a promoter. In certain embodiments, the vehicle is a retrovirus or adenovirus. In certain other embodiments, the nucleic acid molecule is associated with a polycation. In certain other embodiments, the gene delivery vehicle may further comprise a ligand that binds a cell surface receptor.
Turning to another aspect, the invention provides a method of treating cancer by administering to a patient a gene delivery vehicle comprising a nucleic acid molecule encoding a self-oligomerizing caspase-9 polypeptide, wherein the nucleic acid molecule is operatively linked to a promoter, wherein the gene delivery vehicle is internalized by tumor cells. In certain embodiments, the vehicle is a retrovirus or adenovirus. In certain other embodiments, the nucleic acid molecule is associated with a polycation, and in certain other embodiments, the gene delivery vehicle may further comprise a ligand that binds a cell surface receptor.
It is yet another aspect of the invention to provide a method of treating autoimmune disease by administering to a patient a gene delivery vehicle comprising a nucleic acid molecule encoding a self-oligomerizing caspase-9 polypeptide, wherein the nucleic acid molecule is operatively linked to a promoter and wherein the gene delivery vehicle is internalized by tumor cells. In certain embodiments, the vehicle is a retrovirus or adenovirus. In certain other embodiments, the nucleic acid molecule is associated with a polycation.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, the various references set forth below that describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.